3137 fetuses in 33 years: What we have learned from the Wisconsin stillbirth service program.

The Wisconsin Stillbirth Service Program (WiSSP) provided expert review by a dysmorphologist for community-acquired data on 3137 fetal deaths between 1983 and 2017. Intrinsic fetal causes were consistently identified in about 25% while placental and maternal causes were recognized with increasing frequency as attention was shifted from a primarily fetal to a multifocal approach. Identification of causes increased from 40% to 78% and in about half of cases results of the review altered recurrence risk and/or future pregnancy management. Banked data from WiSSP formed the basis of 24 publications, more than half of which have a genetic counselor and/or summer premedical student intern as an author. The earlier publications emphasized validation of the concept of community-based evaluation with central review, the utility of various parts of the WiSSP protocol, the similarity of second-trimester miscarriages <20 weeks to later stillbirths with respect to causes identified and recurrence risks, and the potential for results of etiologic evaluation to influence future prenatal care. The most important recurrent theme, however, was the interaction of intrinsic fetal, placental, and maternal factors in contributing to fetal demise. This implies that, at least in developed nations with available obstetric care, reduction in stillbirth will require careful attention to the myriad of factors contributing to fetal, placental, and maternal well-being.

Fetoplacental ratios in stillbirths and second trimester miscarriages.

An abnormal fetoplacental (F/P) ratio is a risk factor for poor pregnancy outcomes including fetal death, but studies of F/P ratio among stillbirths are limited. In the Wisconsin Stillbirth Service Program cohort of second and third trimester fetal deaths, 1,022 were at ≥24 weeks with data on fetal and placental weight. Comparison with data for viable infants of the same gestational ages (GAs) showed that the F/P ratio increases more rapidly with GA for stillbirths than for viable infants. While placentas of stillborn infants are small at all GA, weights of deceased fetuses are lowest early in the second trimester, becoming nearly normal by term. Excess high F/P ratios are noted at all GAs, increasing toward term, while low ratios are frequent at early gestation but rare near term. Analysis by cause of death shows that F/P ratios are markedly elevated for placental and maternal causes (about 50% above the 90th centile), somewhat elevated for cord accidents, non-hydropic fetal, and unknown causes (about 1/3 above the 90th centile), and variable with 40% below the 10th centile for hydropic stillbirths. Across all causes and GAs, placental weights are more abnormal than fetal weights, suggesting that diminished placental function may contribute to fetal demise even when non-placental causes (e.g., premature membrane rupture, cord accidents, and chromosomal disorders) are identified. About half of all stillbirths have abnormal F/P ratios, suggesting that improvements in prenatal assessment of placental size and function might aid in identifying pregnancies at risk for demise; unfortunately, therapeutic options for ongoing pregnancies with diminished placental function remain limited.

Cardiac Anomalies in Liveborn and Stillborn Monochorionic Twins.

BACKGROUND: Cardiovascular anomalies are more common in monochorionic twins, especially with twin-twin transfusion, compared to other twin types and to singletons. Because previous studies are based on fetal and neonatal echocardiography, more information is needed to study prevalence of cardiac anomalies in twin miscarriages, stillbirths, and children after the immediate neonatal period. METHODS: With specific attention to cardiac anomalies, we reviewed the medical records of 335 selected liveborn twin pairs from the Marshfield Clinic Twin Cohort (enriched for twin-twin transfusion) and all twins (175 pairs) identified in the Wisconsin Stillbirth Service Program cohort of late miscarriages and stillbirths. RESULTS: Structural cardiac defects occurred in 12% of liveborn monochorionic twin infants and 7.5% of stillborn infants with twin-twin transfusion compared to only 2% of liveborn dizygotic twins and no stillborn dizygotic infants. The most common cardiac lesion in liveborn twins was ventricular septal defect, which was usually isolated and discordant, preferentially affecting the smaller twin in monochorionic pairs. Among stillborn and miscarried monochorionic twins, the most common cardiac lesion was acardia. CONCLUSIONS: Monochorionic twins, particularly those with TTT, are at increased risk for a spectrum of structural cardiac malformations which we suggest may be related to asymmetry of the inner cell mass resulting in a smaller poorly perfused twin. In severe cases, limited cardiac and circulatory development in the affected twin leads to acardia. In less severe cases, the smaller infant has deficient septal growth that sometimes results in ventricular septal defect.

Hydrops fetalis in a cohort of 3,137 stillbirths and second trimester miscarriages.

Hydrops fetalis was diagnosed in 277 (9%) of 3,137 fetuses referred to the Wisconsin Stillbirth Service Program (WiSSP) for etiologic evaluation of stillbirth or second trimester miscarriage. Hydrops was clinically recognized at delivery in only about half the cases, while the remainder were diagnosed at autopsy or during evaluation of records, photographs, and radiographs. The peak incidence of hydrops was at 20-28 weeks. Hydropic fetuses were also frequent before 20 weeks but became increasingly rare toward term. The most frequent identifiable underlying cause was chromosomal (29%), followed by other syndromes (14%), and more distantly by cardiac (6%) and other single system disorders. While the overall prevalence of hydrops and chromosomal causes was comparable to other autopsy series, the frequency of nonchromosomal syndromes was higher, reflecting increased attention to syndrome identification. Lethal multiple pterygium syndrome (LMPS) was identified retrospectively in 17 cases, accounting for 6% of all hydrops; 3/17 had a previous affected sib, emphasizing the importance of accurate diagnosis and counseling. Depending on the underlying cause, hydropic fetuses may be either small (if the cause is chromosomal or LMPS) or large (in cases with other syndromes or cardiac causes) for gestational age. The relatively large number in the "idiopathic" group in WiSSP (104/277; 38%) is probably due to variability of autopsies at local hospitals and limited laboratory data. Improved recognition of hydrops and testing directed at diagnosis of specific underlying causes can lead to improved counseling for families.

Early demise of twins in a cohort of stillbirths and second trimester miscarriages.

Twins, particularly monochorionic (MC) pairs, are at increased risk for fetal death. Whereas previous work has sought to understand the mechanisms for this increased mortality, most studies analyze viable twin pregnancies or liveborn twin cohorts. In the Wisconsin Stillbirth Service Program cohort of 3,137 stillbirths and second trimester miscarriages, we identified 175 twin pregnancies for a twinning rate of 56/1,000, which is approximately double the general population. The excess of twins among miscarriages and stillbirths was attributable to MC pairs as the incidence of dizygotic (DZ) twinning was not increased compared to livebirth data. The leading causes of fetal demise among twins were twin-twin transfusion, acardia, and twin-twin disruption. Maternal causes of death, primarily premature rupture of membranes, were moderately increased in both MC and DZ twins relative to singletons. Although deceased twins were smaller than expected for viable twins at comparable gestational ages, placenta weights of deceased MC pairs were large compared to combined fetal weight, which indicates placental inefficiency likely due to vascular shunting. Co-twin survival was much lower for MC than for DZ pairs. Therefore, earlier diagnosis and treatment of MC twinning complications may decrease prenatal mortality.

De novo mutations in NALCN cause a syndrome characterized by congenital contractures of the limbs and face, hypotonia, and developmental delay.

Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal-dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five individuals who had been putatively diagnosed with "DA2A with severe neurological abnormalities" and for whom congenital contractures of the limbs and face, hypotonia, and global developmental delay had resulted in early death in three cases; this is a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in the sodium leak channel, non-selective (NALCN) in four families affected by CLIFAHDD syndrome. We used molecular-inversion probes to screen for NALCN in a cohort of 202 distal arthrogryposis (DA)-affected individuals as well as concurrent exome sequencing of six other DA-affected individuals, thus revealing NALCN mutations in ten additional families with "atypical" forms of DA. All 14 mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that NALCN alterations nearly abolished the expression of wild-type NALCN, suggesting that alterations that cause CLIFAHDD syndrome have a dominant-negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families affected by an autosomal-recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition characterized by varied though overlapping phenotypic features, perhaps based on the type of mutation and affected protein domain(s).

Mutations in PDGFRB cause autosomal-dominant infantile myofibromatosis.

Infantile myofibromatosis (IM) is a disorder of mesenchymal proliferation characterized by the development of nonmetastasizing tumors in the skin, muscle, bone, and viscera. Occurrence within families across multiple generations is suggestive of an autosomal-dominant (AD) inheritance pattern, but autosomal-recessive (AR) modes of inheritance have also been proposed. We performed whole-exome sequencing (WES) in members of nine unrelated families clinically diagnosed with AD IM to identify the genetic origin of the disorder. In eight of the families, we identified one of two disease-causing mutations, c.1978C>A (p.Pro660Thr) and c.1681C>T (p.Arg561Cys), in PDGFRB. Intriguingly, one family did not have either of these PDGFRB mutations but all affected individuals had a c.4556T>C (p.Leu1519Pro) mutation in NOTCH3. Our studies suggest that mutations in PDGFRB are a cause of IM and highlight NOTCH3 as a candidate gene. Further studies of the crosstalk between PDGFRB and NOTCH pathways may offer new opportunities to identify mutations in other genes that result in IM and is a necessary first step toward understanding the mechanisms of both tumor growth and regression and its targeted treatment.

Recurrence of stillbirth and second trimester pregnancy loss.

The 3,003 women referred to the Wisconsin Stillbirth Service Program following a stillbirth or second trimester fetal death reported a total of 4,563 previous pregnancies including 180 previous second or third trimester losses for a total precurrence rate of 3.95%. The 142 women with a history of at least one previous stillbirth and/or late miscarriage differed significantly from the entire cohort with respect to timing and cause of their losses. Women experiencing multiple losses frequently had both second trimester miscarriages and stillbirths >20 weeks but did not have an increased risk of first trimester miscarriage. Recurrences were more likely to be in the second trimester (52% vs. 37%) and to have a maternal (20% vs. 11%) or placental (27% vs. 19%) cause. While fetal causes overall were less common in the group with recurrence (18% vs. 27%), the difference was due mainly to fewer common aneuploidies and other low recurrence risk conditions. Not only known recessive conditions but also "idiopathic hydrops" and multiple congenital anomalies not fitting a known syndrome were more frequent than expected, suggesting that these groups should be investigated for underlying genetic causes that might have been overlooked. Women with second trimester losses and/or a maternal or placental cause of death face significantly higher empiric risks (7-8% vs. 4% for the entire cohort) and should be counseled accordingly. Study of recurrent fetal loss can help identify high risk women who may benefit from treatment and preventive strategies in the future.

Minor anomalies in stillborn and second trimester miscarried fetuses.

In 1964, the landmark paper of Marden, Smith, and McDonald established that multiple minor anomalies in newborn infants are associated with an increased risk for major malformations. There were until now no comparable studies in stillbirths. The Wisconsin Stillbirth Service Program (WiSSP) has data regarding nearly 3,000 stillbirths and second trimester losses that have been analyzed for major anomalies and cause of death. One dysmorphologist retrospectively reviewed all 2,397 with usable photographs. Minor anomalies were identified in 1,413 (59%) with 575 of these (41%) having at least one major anomaly. Probability of a major anomaly increased from 7% with no minor anomalies to 15%, 36%, 67%, and 89% with 1, 2, 3, and >33 minor anomalies, respectively. Frequency of minor anomalies was less with lower resolution photographs, but did not show significant differences with maceration or gestational age. The most frequent minor anomalies were infraorbital creases/folds, lowset/posteriorly angulated ears, nuchal edema, flat face, equinovarus foot, camptodactyly, upslanted palpebral fissures, ear antihelix abnormalities (combined), micrognathia/retrognathia, and single transverse palmar crease. Except for infraorbital creases/folds each of these minor anomalies was strongly correlated with major anomalies (P < 0.0001). Infraorbital folds were the only anomaly which increased with placental cause of death, and reanalysis with placental causes excluded showed the expected relationship to major anomalies, suggesting that infraorbital folds may be markers for oligohydramnios due to various causes including placental hypoperfusion. Minor anomalies correlate with presence of major anomalies in stillborn fetuses, regardless of gestational age and maceration, and can provide information to guide decisions regarding laboratory testing and other evaluations.

SeqHBase: a big data toolset for family based sequencing data analysis.

BACKGROUND: Whole-genome sequencing (WGS) and whole-exome sequencing (WES) technologies are increasingly used to identify disease-contributing mutations in human genomic studies. It can be a significant challenge to process such data, especially when a large family or cohort is sequenced. Our objective was to develop a big data toolset to efficiently manipulate genome-wide variants, functional annotations and coverage, together with conducting family based sequencing data analysis. METHODS: Hadoop is a framework for reliable, scalable, distributed processing of large data sets using MapReduce programming models. Based on Hadoop and HBase, we developed SeqHBase, a big data-based toolset for analysing family based sequencing data to detect de novo, inherited homozygous, or compound heterozygous mutations that may contribute to disease manifestations. SeqHBase takes as input BAM files (for coverage at every site), variant call format (VCF) files (for variant calls) and functional annotations (for variant prioritisation). RESULTS: We applied SeqHBase to a 5-member nuclear family and a 10-member 3-generation family with WGS data, as well as a 4-member nuclear family with WES data. Analysis times were almost linearly scalable with number of data nodes. With 20 data nodes, SeqHBase took about 5 secs to analyse WES familial data and approximately 1 min to analyse WGS familial data. CONCLUSIONS: These results demonstrate SeqHBase's high efficiency and scalability, which is necessary as WGS and WES are rapidly becoming standard methods to study the genetics of familial disorders.